Massive, worldwide efforts over the past few decades have resulted, or will soon result, in the complete genome sequencing of a number of select organisms. Once completed and cataloged into databases, these genomes represent virtual libraries that can be translated into all of the potential proteins contained within their respective organisms. In this regard, genome databases become a foundation for the much broader field of proteomics, wherein the structure and function of specific proteins are under investigation. Such studies are intrinsically complicated, representing multi-dimensional problems that are both qualitative and quantitative in nature. To begin with, a protein of interest will generally reside in complex biological systems and oftentimes represents only a small fraction of the total protein content of the system. The efficient fractionation of the protein from bulk endogenous compounds is therefore necessary for any further characterization.
Once isolated, the protein must be characterized in terms of structure. The focus of these analyses ranges from primary (amino acid sequence) through tertiary (three-dimensional structure due to protein folding) structure, as well as post-translational modifications. Proteome investigation then takes on the new dimension of protein function, with emphasis placed on quaternary structure (polypeptide complexes resulting in functional proteins) and the determination of biomolecular interaction partners (receptor-ligand interactions).
Finally, it is often necessary to quantitatively monitor expression profiles to better understand protein function as a part of a complete cellular system.
In all, it is accurate to say that proteome investigations demand much of analytical sciences and corresponding instrumentation. Thus, there exists a growing need for concerted, multi-analytical approach capable of high-sensitivity analyte fractionation and characterization of protein structure and function.
Multidimensional chip-integrated microarrays that encompass both selective protein fractionation and complete structural/functional characterization would satisfy the need for such proteome analysis using “lab-on-a-chip” approaches. These chip-based microarrays must be combined with a microfluidics system able to precisely deliver a sample to specialized sites on the chip that are capable of analyte fractionation and subsequent processing and/or modifications. The chip based microfluidics systems preferably includes a form of detection for sensing the isolation of an analyte and for tracking the location of the analyte on the array during manipulation. Finally, the system must be capable of providing defining structural information on the analyte, such as sequence verification and/or identification, and detection of point mutations and post-translational modification, thereby resulting in analyte identification.
In 1988, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was introduced by Hillenkamp and Karas (Anal. Chem. 60:2288-2301, 1988), and, has since become a valuable tool for protein characterization and identification. Briefly, MALDI-TOF mass spectrometry is based on the ability to generate intact vapor-phase ions of large, thermally labile biomolecules by desorption/ionization from a matrix comprised of small volatile (matrix) molecules and the biomolecules studied. Pulsed laser radiation, tuned to an absorption maximum of the matrix is used to initiate the desorption/ionization event and to simultaneously generate a packet of ions of different mass-to-charge ratio (m/z). These ions are accelerated to the same electrostatic potential and allowed to drift an equal distance before striking a detector. The mass of the ions is determined by equating the flight times of the ions to m/z.
The success of MALDI-TOF mass spectrometry in the area of protein science can be attributed to several factors. The greatest of these is that MALDI-TOF can be rapidly (˜5 minutes) applied as an analytical technique to analyze small quantities of virtually any protein (practical sensitivities of ˜1 pmole protein loaded into the mass spectrometer). The technique is also extremely accurate in identifying biomolecules. Beavis and Chait demonstrated that the molecular masses of peptides and proteins can be determined to within 0.01% by using methods in which internal mass calibrants (x-axis calibration) are introduced into the analysis (Anal. Chem. 62:1836-40, 1990).
MALDI-TOF can also be quantitatively used with a similar method in which internal reference standards are introduced into the analysis for ion signal normalization (y-axis calibration) with accuracies on the order of 10% (Nelson et al., Anal. Chem. 66:1408-15, 1994).
Finally, MALDI-TOF is extremely tolerant of large molar excesses of buffer salts and, more importantly, the presence of other proteins, making it a practical approach for directly analyzing complex biological mixtures. Many examples exist where MALDI-TOF is used to directly analyze the results of proteolytic or chemical degradation of polypeptides. Other examples extend to elucidating post-translational modifications (namely carbohydrate type and content), a process requiring the simultaneous analysis of components present in a heterogeneous glycoprotein mixture (Sutton et al., Techniques in Protein Chemistry III, Angeletti, Ed., Academic Press, Inc., New York, pp. 109-1 16, 1993). Arguably, the most impressive use of direct mixture analysis is the screening of natural biological fluids. In this application, proteins are identified, as prepared directly from the host fluid, by detection at precise and characteristic mass-to-charge (m/z) values (Tempst et al., Mass Spectrometry in the Biological Sciences, Burlingame and Carr, Ed., Humana Press, Totowa, N.J., p. 105, 1996).
While the above examples involving direct MALDI-TOF analysis of complex mixtures are quite impressive, there exist limits to the extent of practical application. These limits are reached when a target analyte is a minor component of the mixture, and is present at low concentration. A common occurrence in such situations is that the target analyte is never observed in the MALDI-TOF mass spectrum. This lack of detection is generally due to the low concentration of the analyte, yielding ion signals at or below the instrumental limits of detection. This effect is further exacerbated by protein-analyte interactions “stealing” analyte molecules from the MALDI-TOF process and/or a high instrumental baseline produced from other proteins present in the mixture resulting in “analyte masking”. Methods for the selective concentration of specific species in the mixture were therefore required in order to achieve ion signals from the target analyte.
Biomolecular Interaction Analysis (BIA) is a technique capable of monitoring interactions, in real time and without the use of labels, between two or more molecules such as proteins, peptides, nucleic acids, carbohydrates, lipids and low molecular weight molecules (i.e. signaling substances and pharmaceuticals). Molecules do not need to be purified or even solubilized for BIA, but can be studied in crude extracts as well as anchored in lipid vesicles, viruses, bacteria and eucaryotic cells. In one form, the detection of molecular interactions in BIA is via Surface Plasmon Resonance (SPR), taking form of SPR-BIA. The detection principle of SPR-BIA relies on the optical phenomenon of SPR, which detects changes in the refractive index of the solution close to the surface of a sensor device, or chip. An SPR sensor consists of a transparent material having a metal layer deposited thereon. One of the interactants (e.g., receptor) is immobilized on the metal surface layer of the sensor that forms one wall of a micro-action site. A light source generates polarized light that is directed through a prism, or diffraction grating, striking the metal layer-transparent material interface. A detector detects light reflected from the metal surface. A sample containing the other interaction partner is injected in a controlled flow over the surface containing the bound interactant. Any change in the surface concentrations resulting from an interaction between the two, or more, interactants, is spectroscopically detected as an SPR signal by the shifting of relative reflective intensity signals. A continuous display of the SPR signal, as a function of time, yields a “sensorgram” that provides a complete record of the progress of associations and disassociations. When analysis of one interaction cycle is completed, the surface of the sensor can be regenerated by treatment with conditions that remove all bound analytes without affecting the activity of the immobilized ligand. SPR-BIA has become a valuable tool for the functional characterization of proteins and is broadly used for determining the kinetic and affinity parameters involved in biomolecular interactions; however, no structural information on the interacting biomolecules can be gained from the SPR-BIA analysis.
In a recent invention, SPR-BIA and MALDI-TOF MS were combined in a method that allows selective retrieval and retention of an analyte from solution on a sensor chip (monitored by SPR-BIA), followed by MALDI-TOF MS analysis of the sensor chip, yielding the mass of the retained analyte (see U.S. Pat. No. 5,955,729 entitled “Surface Plasmon Resonance-Mass Spectrometry (SPR-MS)”. Thereby, this method performs a selective concentration of specific analytes from the analyzed mixture in quantities sufficient to achieve analyte ion signals for MALDI-TOF MS. Whereas the SPR-MS combination represents an important improvement over the SPR-BIA analysis alone and allows for functional concentration of a targeted analyte (important for MALDI-TOF MS analysis of low-level analytes), both SPR-BIA and SPR-MS have been limited to analyzing biomolecules as they exist in the solution, which, in many instances, can not provide enough information for the biomolecules identification. Frequently, in order to fully characterize and identify certain biomolecules, modification or derivatization is required.
Thus, there is a need for devices and methods that allow for the isolation of small quantities of biomolecules from a complex solution and that are able to modify, or bioreact, these biomolecules to create characteristic fragments, derivatives and the like. Accordingly, a variety of chemistries, enzymologies, characterizations and identification techniques may be employed after analyte isolation to gain information on both original and reacted biomolecules. Importantly, these chemistries, enzymologies, characterizations and identification techniques must be performed in manners that do not introduce interfering artifacts, e.g., backgrounds arising from the modifying reagents, the immobilized receptor responsible for isolating the analyte or from instrumental changes, into the analysis. Therefore, it is preferred to perform these characterizing modifications using: immobilized reagents, such as enzymes and the like (to preclude the introduction of reagent-induced artifacts), which are present at different sites than the receptor (such as not to introduce artifacts due to the receptor), and that analyses are performed on the same platform (such as to minimize instrumental changes). The present invention fulfills these needs, and provides further related advantages